Jim R. Stockigt

Non‐isotopic spectrophotometric determination of the unbound fraction of drugs in serum*

A spectrophotometric method to measure the free fraction of highly‐bound drugs in serum has been established for a range of non‐steroidal anti‐inflammatory drugs (NSAIDs) and for frusemide. Spectrophotometry is used to measure fractional transit of drug from a large volume of dialysate to a small volume of serum during dialysis to equilibrium. The method, which depends on the principle that drug transit from dialysate to serum is proportional to serum binding, requires neither isotopic drug preparations nor specific drug assays, is independent of extraction efficiency from the dialysate and requires no measurements from the serum compartment. Estimates of percent unbound fraction (%UF) for aspirin (6.0 ± 0.9%), phenylbutazone (0.9 ± 0.2%), and frusemide (1.8 ± 0.2%) were comparable with those obtained with 14C drug preparations. Values for %UF were determined for eleven additional NSAIDs. The method was valid for a four‐fold change in serum: dialysate ratio. Kinetics of frusemide binding to serum were comparable using [14C]frusemide and the test method. This technique may have general application in establishing the %UF for substances that are extensively bound to serum proteins and for identifying sera that show abnormal binding.

The 4-mg intravenous dexamethasone suppression test in the diagnosis of Cushing’s syndrome

Objective  Optimal diagnostic criteria for the 4‐mg intravenous dexamethasone suppression test (IVDST) in patients with Cushing’s syndrome (CS), compared with normal subjects, have not been established. We evaluated the performance of the 4‐mg IVDST for differentiating CS from normal subjects and to define the responses in CS of various aetiologies.

Anomalous binding characteristics of human thyroxine binding globulin due to a dilution-dependent separation artefact

Contrary to the accepted view, a recent study using Sephadex column separation suggested that thyroxine binding globulin (TBG) binds T4 and T3 with similar affinity, but with a much larger capacity for T4 than T3. We have evaluated this finding by comparing this separation method with equilibrium dialysis, taking account of the effect of serum dilution with each method. Estimates of free T4 fraction by equilibrium dialysis (with magnesium chloride precipitation) were valid over a wide range of serum dilutions. In contrast, Sephadex column separation gave a major overestimate of free hormone (underestimate of binding) in less diluted serum, indicating that this method cannot be used to establish a value for T4 affinity independent of serum dilution. Such a systematic error will result in a greater underestimate of affinity for the ligand with higher affinity when two ligands are compared at a single serum dilution. By equilibrium dialysis at 37°C, the affinity of T4 for TBG was ∼13‐fold higher than that of T3, while the capacity of TBG for both T4 and T3 was close to the concentration of immunoreactive TBG. The previous report of similar T4 and T3 affinities appears to be due to a dilution‐dependent underestimate of T4 affinity inherent in Sephadex column separation. Direct comparison of binding kinetics of various ligands requires a separation method that is valid over a wide range of binding protein concentrations.

Measurements of Thyroxine and Triiodothyronine

The serum concentrations of thyroxine (T4) and triiodothyronine (T3) are key parameters in the assessment of thyroid function. As a thyroid disorder progresses from normality to overt dysfunction, the concentration of thyroid stimulating hormone (TSH) generally falls outside the population “normal” range before the concentration of either T4 or T3 becomes diagnostic, but a twofold change in serum T4 generally correlates better with clinical features than a twofold change in TSH. When thyroid dysfunction is corrected, the return of TSH to normal may lag some months behind the clinical response. Thus, the severity of the disorder and the initial response to treatment are often best monitored by measurement of the end-organ products, T4 and T3. Further, the diagnosis of secondary, or central hypothyroidism is likely to be missed unless end-organ function is assessed by measurement of T4. The limitations of basing initial testing for thyroid dysfunction on TSH alone have been discussed elsewhere [1, 2].

Drug Effects and Thyroid Function

A wide range of therapeutic agents cause unintended alterations in the pituitary–thyroid axis. Some drugs can directly alter thyroid function and numerous other medications can produce apparent abnormalities in standard tests of thyroid function, especially during illness. Familiarity with these effects is necessary to distinguish the transient or spurious effects of medications from spontaneous abnormalities.